title: Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins.
abstract: Leishmania tarentolae is a non-human-pathogenic Leishmania species of growing interest in biotechnology, as it is well-suited for the expression of human recombinant proteins. For many applications it is desirable to express recombinant proteins with a tag allowing easy purification and detection. Hence, we adopted a scheme to express recombinant proteins with a His6-tag and, additionally, to site-specifically in vivo biotinylate them for detection. Biotinylation is a relatively rare modification of endogenous proteins that allows easy detection with negligible cross-reactivity. Here, we established a genetically engineered L. tarentolae strain constitutively expressing the codon-optimized biotin-protein ligase from Escherichia coli (BirA). We thoroughly analyzed the strain for functionality using 2-D polyacrylamide-gel electrophoresis (PAGE), mass spectrometry, and transmission electron microscopy (TEM). We could demonstrate that neither metabolic changes (growth rate) nor structural abnormalities (TEM) occurred. To our knowledge, we show the first 2-D PAGE analyses of L. tarentolae. Our results demonstrate the great benefit of the established L. tarentolae in vivo biotinylation strain for production of dual-tagged recombinant proteins. Additionally, 2-D PAGE and TEM results give insights into the biology of L. tarentolae, helping to better understand Leishmania species. Finally, we envisage that the system is transferable to human-pathogenic species.
authors.affiliation: Max Planck Institute for Molecular Genetics , Ihnestr. 63-73, 14195 Berlin, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Klatt
authors.forename: Daniela
authors.initials: D
authors.lastname: Hartl
authors.forename: Beatrix
authors.initials: B
authors.lastname: Fauler
authors.forename: Dejan
authors.initials: D
authors.lastname: Gagoski
authors.forename: Susana
authors.initials: S
authors.lastname: Castro-Obregón
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Konthur
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001181:Arthropods / Q000469:parasitology; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism; D002853:Chromatography, Liquid; D003062:Codon; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D019076:Transgenes
chemical_list: D003062:Codon; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli